Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

Christopher P Stefan,Adrienne T Hall,Timothy D Minogue

doi:10.1038/s41598-018-19501-z

Abstract

Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.

Highlights

Analysis of the 16S sequence revealed that only specific hypervariable regions, in particular variable regions V2, V3, and V6 together, were required for identification of common bacterial pathogens[9,10]
next-generation sequencing (NGS) platforms such as Illumina produce read lengths smaller than those required for full length 16S gene sequencing or for amplicons that include the number of variable regions required for low-level taxonomic resolution[10,11]
Molecular Inversion probes (MIP) are one method to enrich for target sequences using a ligation dependent approach, which allows for a high order of multiplexabiltiy within a single tube reaction[14,15]

Summary

Introduction

Analysis of the 16S sequence revealed that only specific hypervariable regions, in particular variable regions V2, V3, and V6 together, were required for identification of common bacterial pathogens[9,10]. Given the sequencing depth afforded by NGS, a multiplexed targeted assay could capture and amplify multiple, short length, hypervariable 16S regions along with AR genes, SNPs, and toxin producing elements for identification and characterization. Multiple technologies exist for multiplexed upfront target enrichment of DNA samples prior to NGS including PCR, ligation dependent amplification, and hybridization capture (reviewed in[12]). MIPs involve target-specific sequence hybridization combined with a “gap-fill” step via polymerase and a subsequent ligation event. These dual enzymatic steps are required for proper capture of the targeted region. Combined with the removal of excess probe and unwanted amplification events via exonuclease reaction, this method allows for high-level multiplexing unattainable with other methods. Demonstrated here is the use of MIPs for pathogen identification and AR detection directly after blood culture

Methods

Results

Conclusion